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Artykuły w czasopismach na temat "CHO HOST CELL PROTEINS"
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Hogwood, Catherine EM, Daniel G. Bracewell i C. Mark Smales. "Measurement and control of host cell proteins (HCPs) in CHO cell bioprocesses". Current Opinion in Biotechnology 30 (grudzień 2014): 153–60. http://dx.doi.org/10.1016/j.copbio.2014.06.017.
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Van Manen-Brush, Kathleen, Jacob Zeitler, John R. White, Paul Younge, Samantha Willis i Marisa Jones. "Improving Chinese hamster ovary host cell protein ELISA using Ella®: an automated microfluidic platform". BioTechniques 69, nr 3 (wrzesień 2020): 186–92. http://dx.doi.org/10.2144/btn-2020-0074.
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Chinese hamster ovary (CHO) cells are a mammalian cell line used in the production of therapeutic proteins. Host cell proteins (HCPs) are process-related impurities that are derived from the host cell expression system. During biopharmaceutical drug development, removal of HCPs is required. Enzyme-linked immunosorbent assay (ELISA) is a common technique to quantitate HCPs, but is a labor-intensive process that takes up to 7 h. Ella® is an automated instrument that utilizes microfluidics and glass nanoreactors to quantitate HCPs in 75 min using similar ELISA reagents. The antibodies and antigens are captured on three distinct glass nanoreactors, resulting in sensitive reproducible data. Our results indicate that Ella quantitates CHO HCPs with precision, accuracy, sensitivity and trends comparable with our traditional CHO HCP ELISA.
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Gilgunn, Sarah, i Jonathan Bones. "Challenges to industrial mAb bioprocessing—removal of host cell proteins in CHO cell bioprocesses". Current Opinion in Chemical Engineering 22 (grudzień 2018): 98–106. http://dx.doi.org/10.1016/j.coche.2018.08.001.
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Glinšek, Katja, Krištof Bozovičar i Tomaž Bratkovič. "CRISPR Technologies in Chinese Hamster Ovary Cell Line Engineering". International Journal of Molecular Sciences 24, nr 9 (2.05.2023): 8144. http://dx.doi.org/10.3390/ijms24098144.
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The Chinese hamster ovary (CHO) cell line is a well-established platform for the production of biopharmaceuticals due to its ability to express complex therapeutic proteins with human-like glycopatterns in high amounts. The advent of CRISPR technology has opened up new avenues for the engineering of CHO cell lines for improved protein production and enhanced product quality. This review summarizes recent advances in the application of CRISPR technology for CHO cell line engineering with a particular focus on glycosylation modulation, productivity enhancement, tackling adventitious agents, elimination of problematic host cell proteins, development of antibiotic-free selection systems, site-specific transgene integration, and CRISPR-mediated gene activation and repression. The review highlights the potential of CRISPR technology in CHO cell line genome editing and epigenetic engineering for the more efficient and cost-effective development of biopharmaceuticals while ensuring the safety and quality of the final product.
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Keysberg, Christoph, Oliver Hertel, Louise Schelletter, Tobias Busche, Chiara Sochart, Jörn Kalinowski, Raimund Hoffrogge, Kerstin Otte i Thomas Noll. "Exploring the molecular content of CHO exosomes during bioprocessing". Applied Microbiology and Biotechnology 105, nr 9 (maj 2021): 3673–89. http://dx.doi.org/10.1007/s00253-021-11309-8.
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Abstract In biopharmaceutical production, Chinese hamster ovary (CHO) cells derived from Cricetulus griseus remain the most commonly used host cell for recombinant protein production, especially antibodies. Over the last decade, in-depth multi-omics characterization of these CHO cells provided data for extensive cell line engineering and corresponding increases in productivity. However, exosomes, extracellular vesicles containing proteins and nucleic acids, are barely researched at all in CHO cells. Exosomes have been proven to be a ubiquitous mediator of intercellular communication and are proposed as new biopharmaceutical format for drug delivery, indicator reflecting host cell condition and anti-apoptotic factor in spent media. Here we provide a brief overview of different separation techniques and subsequently perform a proteome and regulatory, non-coding RNA analysis of exosomes, derived from lab-scale bioreactor cultivations of a CHO-K1 cell line, to lay out reference data for further research in the field. Applying bottom-up orbitrap shotgun proteomics and next-generation small RNA sequencing, we detected 1395 proteins, 144 micro RNA (miRNA), and 914 PIWI-interacting RNA (piRNA) species differentially across the phases of a batch cultivation process. The exosomal proteome and RNA data are compared with other extracellular fractions and cell lysate, yielding several significantly exosome-enriched species. Key points • First-time comprehensive protein and miRNA characterization of CHO exosomes. • Isolation protocol and time point of bioprocess strongly affect quality of extracellular vesicles. • CHO-derived exosomes also contain numerous piRNA species of yet unknown function.
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Alhuthali, Sakhr, i Cleo Kontoravdi. "Population balance modelling captures host cell protein dynamics in CHO cell cultures". PLOS ONE 17, nr 3 (23.03.2022): e0265886. http://dx.doi.org/10.1371/journal.pone.0265886.
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Monoclonal antibodies (mAbs) have been extensively studied for their wide therapeutic and research applications. Increases in mAb titre has been achieved mainly by cell culture media/feed improvement and cell line engineering to increase cell density and specific mAb productivity. However, this improvement has shifted the bottleneck to downstream purification steps. The higher accumulation of the main cell-derived impurities, host cell proteins (HCPs), in the supernatant can negatively affect product integrity and immunogenicity in addition to increasing the cost of capture and polishing steps. Mathematical modelling of bioprocess dynamics is a valuable tool to improve industrial production at fast rate and low cost. Herein, a single stage volume-based population balance model (PBM) has been built to capture Chinese hamster ovary (CHO) cell behaviour in fed-batch bioreactors. Using cell volume as the internal variable, the model captures the dynamics of mAb and HCP accumulation extracellularly under physiological and mild hypothermic culture conditions. Model-based analysis and orthogonal measurements of lactate dehydrogenase activity and double-stranded DNA concentration in the supernatant show that a significant proportion of HCPs found in the extracellular matrix is secreted by viable cells. The PBM then served as a platform for generating operating strategies that optimise antibody titre and increase cost-efficiency while minimising impurity levels.
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Santoso, Adi. "PERKEMBANGAN TEKNOLOGI SEL MAMALIA CHINESE HAMSTER OVARY (CHO) UNTUK PRODUKSI OBAT BERBASIS PROTEIN". BERITA BIOLOGI 18, nr 2 (27.08.2019): 125–33. http://dx.doi.org/10.14203/beritabiologi.v18i2.3705.
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Chinese hamsters ovary (CHO) and its derivative such as CHO-DXB11 cells, CHO-K1, CHO-DG44 and CHO-S are mammalian cells that are often used for production of therapeutic protein drugs. The CHO cells often used for protein production have several advantages including 1) host cells that are safe to use in drug production, 2) the level of production of proteins produced can be increased by amplifying genes using methotrexate (MTX), 3) having the capacity to make post-translation modificationsand 4) CHO cells can be adapted to grow in suspension. The high need for protein-based drugs triggers the development of basic knowledge and innovation in production of recombinant proteins. The impressive technological advances in CHO cell technology have made these cells can be used to produce proteins around 10 g/liter in order to meet the market demand. The first protein successfully produced using CHO mammalian cells was the therapeutic Tissue Plasminogen Activator (r-tPA, Activase) protein used for stroke patients. The presence of this drug is quickly followed by several other types of drugs. In this review, history of development of CHO cells, the contribution of CHO cells to basic research, progress of effective line cell screening and development technology are discussed.
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Romanova, Nadiya, Louise Schelletter, Raimund Hoffrogge i Thomas Noll. "Hyperosmolality in CHO cell culture: effects on the proteome". Applied Microbiology and Biotechnology 106, nr 7 (21.03.2022): 2569–86. http://dx.doi.org/10.1007/s00253-022-11861-x.
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AbstractChinese hamster ovary (CHO) cells are the most commonly used host cell lines for therapeutic protein production. Exposure of these cells to highly concentrated feed solution during fed-batch cultivation can lead to a non-physiological increase in osmolality (> 300 mOsm/kg) that affects cell physiology, morphology, and proteome. As addressed in previous studies (and indeed, as recently addressed in our research), hyperosmolalities of up to 545 mOsm/kg force cells to abort proliferation and gradually increase their volume—almost tripling it. At the same time, CHO cells also show a significant hyperosmolality-dependent increase in mitochondrial activity. To gain deeper insight into the molecular mechanisms that are involved in these processes, as detailed in this paper, we performed a comparative quantitative label-free proteome study of hyperosmolality-exposed CHO cells compared with control cells. Our analysis revealed differentially expressed key proteins that mediate mitochondrial activation, oxidative stress amelioration, and cell cycle progression. Our studies also demonstrate a previously unknown effect: the strong regulation of proteins can alter both cell membrane stiffness and permeability. For example, we observed that three types of septins (filamentous proteins that form diffusion barriers in the cell) became strongly up-regulated in response to hyperosmolality in the experimental setup. Overall, these new observations correlate well with recent CHO-based fluxome and transcriptome studies, and reveal additional unknown proteins involved in the response to hyperosmotic pressure by over-concentrated feed in mammalian cells.Key points• First-time comparative proteome analysis of CHO cells exposed to over-concentrated feed.• Discovery of membrane barrier-forming proteins up-regulation under hyperosmolality.• Description of mitochondrial and protein chaperones activation in treated cells.
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Lavoie, R., Alice di Fazio, R. Blackburn, Michael Goshe, Ruben Carbonell i Stefano Menegatti. "Targeted Capture of Chinese Hamster Ovary Host Cell Proteins: Peptide Ligand Discovery". International Journal of Molecular Sciences 20, nr 7 (8.04.2019): 1729. http://dx.doi.org/10.3390/ijms20071729.
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The growing integration of quality-by-design (QbD) concepts in biomanufacturing calls for a detailed and quantitative knowledge of the profile of impurities and their impact on the product safety and efficacy. Particularly valuable is the determination of the residual level of host cell proteins (HCPs) secreted, together with the product of interest, by the recombinant cells utilized for production. Though often referred to as a single impurity, HCPs comprise a variety of species with diverse abundance, size, function, and composition. The clearance of these impurities is a complex issue due to their cell line to cell line, product-to-product, and batch-to-batch variations. Improvements in HCP monitoring through proteomic-based methods have led to identification of a subset of “problematic” HCPs that are particularly challenging to remove, both at the product capture and product polishing steps, and compromise product stability and safety even at trace concentrations. This paper describes the development of synthetic peptide ligands capable of capturing a broad spectrum of Chinese hamster ovary (CHO) HCPs with a combination of peptide species that allow for advanced mixed-mode binding. Solid phase peptide libraries were screened for identification and characterization of peptides that capture CHO HCPs while showing minimal binding of human IgG, utilized here as a model product. Tetrameric and hexameric ligands featuring either multipolar or hydrophobic/positive amino acid compositions were found to be the most effective. Tetrameric multipolar ligands exhibited the highest targeted binding ratio (ratio of HCP clearance over IgG loss), more than double that of commercial mixed-mode and anion exchange resins utilized by industry for IgG polishing. All peptide resins tested showed preferential binding to HCPs compared to IgG, indicating potential uses in flow-through mode or weak-partitioning-mode chromatography.
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Vallis, Amy J., Viviane Finck-Barbançon, Timothy L. Yahr i Dara W. Frank. "Biological Effects of Pseudomonas aeruginosa Type III-Secreted Proteins on CHO Cells". Infection and Immunity 67, nr 4 (1.04.1999): 2040–44. http://dx.doi.org/10.1128/iai.67.4.2040-2044.1999.
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ABSTRACT A strain of Pseudomonas aeruginosa that fails to express known type III-secreted effector proteins was constructed as an expression host. Individual effectors were expressed intrans, and their biological effects on CHO cells were assessed in an acute cellular infection model. Intoxication with ExoS, ExoT, or ExoY resulted in alterations in cell morphology. As shown in previous genetic studies, ExoU expression was linked to acute cytotoxicity.
Rozprawy doktorskie na temat "CHO HOST CELL PROTEINS"
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Goey, Cher Hui. "Cascading effects in bioprocessing : the impact of cell culture environment on CHO cell behaviour and host cell protein species". Thesis, Imperial College London, 2016. http://hdl.handle.net/10044/1/52703.
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One of the reasons for the rejection of new drugs during clinical trials is the presence of host cell proteins (HCPs) in the drug formulation. HCPs are immunogenic impurities that can compromise patient safety. Moreover, proteolytic and binding HCPs compromise the integrity, and, hence, the stability and efficacy of a recombinant protein. Therefore, HCPs should be removed from the bioprocess train as soon as possible. Current downstream purification platforms are challenged by HCP-mAb co-elution. A Quality by Design strategy to overcome this problem is to reduce the number of HCPs in the downstream feedstock by tracing their source back to upstream culture and eliminating it. Previous studies have shown that upstream cell culture parameters, e.g. harvest time and culture temperature, significantly affect HCP profiles. However, little is known about how host cells coordinate and regulate their molecular machinery under different cell culture environment that results in different HCP profiles. This study presents experimental results linking cell culture temperature and key process indicators of CHO cell cultures and post-Protein A purification (mAb titre, HCP level and HCP species) by considering the cellular behaviour in terms of cell growth, cell cycle distribution and cell health). This study involved the application of single-use fed-batch bioreactors to culture IgG4 producing GS-CHO cell line, cell health and cell cycle analysis with NucleoCounter, Protein A purification and proteomic analysis with HCP ELISA kits and LC-MS/MS. Cells were more robust under mild hypothermia: over 90% of cells were maintained in a healthy state until the decline phase, and the onset of apoptosis was less evident compared to the results for physiological temperature. IgG4 titre and HCP level at harvest were comparable between the two cases. However, mild hypothermia reduced the HCP variety in HCCF by 36%, including 44% and 27% lower proteases and chaperones, respectively. The differences in HCP variety at harvest resulted in a significantly different HCP profile post-Protein A purification between the two cases. Half of the critically immunogenic HCPs species as determined by the CHOPPI tool were different between the two cases. This study shows that cell culture conditions significantly affect the HCP profile at harvest and that of purified samples.
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Carr, Sharon. "Adenovirus and its interaction with host cell proteins /". St Andrews, 2007. http://hdl.handle.net/10023/219.
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Qashqari, Fadi Saleh I. "Regulation of host cell proteins by adenovirus oncoproteins". Thesis, University of Birmingham, 2017. http://etheses.bham.ac.uk//id/eprint/8020/.
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Adenovirus early region proteins, ElA, E1B-55K, E4orf3 and E4orf6 regulate host cell processes to facilitate viral replication. E4orf3 suppress host cell anti-viral activities through association with host cell proteins in E4orf3 nuclear-track structures, whilst E1B-55K, E4orf3 and E4orf6 are all recruited to viral replication centres during infection to promote viral DNA replication and inhibit host cell antiviral activities. Immunoprecipitation coupled to mass spectrometry identified Toplla as an Ad12 E4orf3-binding protein that localized with E4orf3 in adenovirus-infected cells. It was determined that Toplla expression was induced during infection, and that Toplla was required for the adenovirusdependent stabilization of p53. It was also established that, despite their ability to cooperate functionally, Toplla and p53 do not associate physically during infection. Immunoprecipitation coupled to mass spectrometry was also used to identify host cell proteins recruited to viral replication centres during adenovirus infection. The RP A-1 binding protein, Smarcall, and the FACT complex histone chaperone protein, SSRPl were identified as host cell proteins recruited to viral replication centres during infection. Following recruitment to viral replication centres Smarcall was found to be degraded in an E1B-55K and E4orf6 dependent manner, whilst SSRPl was found to be stable during infection and was not targeted for degradation.
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Bjertsjö, Rennermalm Anna. "Staphylococcal cell wall associated proteins : characteristics and host interactions /". Stockholm, 2005. http://diss.kib.ki.se/2005/91-7140-542-9/.
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Huang, Edwin P. C. Biotechnology & Biomolecular Sciences Faculty of Science UNSW. "Recombinant protein production utilising a metallothionein expression system and a Super-CHO cell line". Awarded by:University of New South Wales. School of Biotechnology and Biomolecular Sciences, 2006. http://handle.unsw.edu.au/1959.4/24940.
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A novel metal-inducible and amplifiable metallothionein (MT) expression system, pNK, was firstly optimised and characterised for the production of a reporter protein, human growth hormone (hGH) in a suspension CHO cell line grown in a serum-free media. The pNK-based hGH production was demonstrated in cadmium-free condition under various fermentation modes (batch, fed-batch and perfusion) and scales (flask to bench-top bioreactor). Improvement of specific productivity of recombinant protein from pNK was shown to be possible by addition of butyrate or substrate substitution of glutamine by glutamate. Combination of fed-batch and butyrate addition strategies resulted in more than one gram per litre of hGH being obtained from the pNK expression system in a bioreactor. In the second part of the project, based on a statistical approach suggested by Plackett-Burman (P-B), a chemically-defined and protein-free medium, named Super-CHO protein-free (SPF), was developed to support a Super-CHO cell line, C2.8-325, to grow as a single-cell suspension culture with comparable growth rate and viable cell number as observed in a commercial medium containing undefined additives. Using Dulbecco's Modified Eagle's Medium/Ham's F12 1:1 mixture (DMEM/F12) as the basal medium, a P-B design matrix screened 10 nutritional components. Components shown potentially beneficial for cell growth rate and viable cell number were supplemented to DMEM/F12 to formulate the SPF medium. Finally, the pNK expression system and the Super-CHO cell line were applied simultaneously in an attempt to express a humanised anti-CD48 monoclonal antibody (MAb), IgG1-N2A (N2A-MAb). This aimed to test C2.8-SPF grown in newly developed SPF medium for transfection, clone development and recombinant protein production. A stable and N2A-MAb expressing C2.8-SPF cell line was successfully constructed, and N2A MAb expression was subsequently amplified and demonstrated in various cultivation scales (flask and bioreactor). This project demonstrated that the novel metal-inducible and - amplifiable mammalian expression system, pNK, and the novel mammalian host cell-line, Super-CHO C2.8-SPF, capable of growing as a single-cell suspension culture in a chemically-defined protein-free medium, SPF, could be utilised in combination to provide a new, low-cost, and regulatory-compliant recombinant protein expression platform, suitable for the biopharmaceutical industry to use in the manufacture of therapeutic recombinant proteins.
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Mohamed, Ahmed Attia Ali. "Interaction of hepatitis C virus polymerase with host cell proteins". Thesis, Durham University, 2009. http://etheses.dur.ac.uk/2107/.
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Hepatitis C virus (HCV) interacts with host cell proteins to modify cellular pathways creating a favourable environment that facilitates its replication and persistence. The purpose of the work presented in this thesis was to identify cellular proteins that can interact with NS5B, the virus's RNA-dependent RNA polymerase, that may contribute to the virus's biology. A number of cellular proteins were found to interact with NS5B using the yeast two-hybrid system. These proteins were involved in cellular pathways such as interferon signalling, lipid transport and metabolism, protein trafficking, cell proliferation and apoptosis. Of these, phospholipid scramblase 1 (PLSCR1) and zinc finger protein 143 (ZNF143) were selected for further investigation. The interactions were confirmed in vitro, and, for PLSCR1, the region that interacted with NS5B was determined to be within the amino-terminal region of the protein (61-137 a.a.). NS5B interacted with PLSCR1 and ZNF143 via a single interacting region localized in its N-terminus (1-153 a.a.).Expression of PLSCR1 or ZNF143 enhanced the ability of interferon to stimulate transcription from an interferon-stimulated response element (ISRE) reporter construct. Co-expression with NS5B was found to down-regulate this activity. Expression of a number of interferon-stimulated genes was investigated in the presence of NS5B, PLSCR1 or ZNF143 but no significant effect was observed. Overexpression of PLSCR1 had no effect on HCV sub-genomic replicon replication, while reduction of its expression by short hairpin RNA (shRNA) enhanced replication. Overexpression of ZNF143 was found to have a suppressive effect on replication but downregulating its expression did not enhance replication. In addition to using the yeast two-hybrid system to identify NS5B- interacting proteins, an in vitro pulldown assay coupled with mass spectrometry identified α- and β -tubulin associated with NS5B in vitro and in vivo. Subsequently this association was demonstrated to be an indirect interaction but the intermediatory partner was not identified. The domain that mediated the association with α- and β-tubulin was determined to be within the N-terminus of NS5B (1-153 a.a.). Nocodazole, an inhibitor of tubulin polymerization, had a marked effect on the association of α -tubulin with NS5B displacing it from the complex but had no effect on β -tubulin's association. Utilizing an HCV sub- genomic replicon, nocodazole was shown to have a significant inhibitory effect on replication. Taken together the data presented in this thesis showed that NS5B had a multitude of potential interactions with a variety of cellular proteins. The biological significance of some of these interactions on the cellular response to IFN and replicon replication was investigated. This work has generated a number of novel observations on the interaction between the virus and the cell that warrant future investigation
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Goldman, Merlin Hesper. "The effect of process variables on the glycosylation of gamma-interferon produced in CHO cells". Thesis, University College London (University of London), 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.244743.
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Patel, Amit. "Interaction of enteropathogenic 'Escherichia coli' (EPEC) tir with host cell proteins". Thesis, University of Nottingham, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.431869.
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Cowlishaw, Deborah Anne. "Identification of host proteins required for bacteriophage infection of Streptomyces sp". Thesis, University of Nottingham, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.367589.
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Rytkönen, Anne. "Molecular studies of Neisseria - host cell interactions /". Stockholm, 2004. http://diss.kib.ki.se/2004/91-7140-018-4/.
Pełny tekst źródłaKsiążki na temat "CHO HOST CELL PROTEINS"
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Spearman, Paul, i Eric O. Freed, red. HIV Interactions with Host Cell Proteins. Berlin, Heidelberg: Springer Berlin Heidelberg, 2009. http://dx.doi.org/10.1007/978-3-642-02175-6.
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O, Freed Eric, i SpringerLink (Online service), red. HIV Interactions with Host Cell Proteins. Berlin, Heidelberg: Springer-Verlag Berlin Heidelberg, 2010.
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1942-, Cabello Felipe C., Pruzzo Carla i North Atlantic Treaty Organization. Scientific Affairs Division., red. Bacteria, complement, and the phagocytic cell. Berlin: Springer-Verlag, 1988.
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L, Crowell Richard, Lonberg-Holm Karl i American Society for Microbiology, red. Virus attachment and entry into cells: Proceedings of an ASM conference held in Philadelphia, Pennsylvania, 10-13 April 1985. Washington, D.C: American Society for Microbiology, 1986.
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Jean-Pierre, Gorvel, red. Intracellular pathogens in membrane interactions and vacuole biogenesis. Georgetown, Tex: Landes Bioscience, 2004.
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M, McCrae, i Society for General Microbiology, red. Molecular aspects of host-pathogen interaction: Fifty-fifth Symposium of the Society for General Microbiology : held at Heriot-Watt University, Edinburgh, March 1997. Cambridge: Cambridge University Press, 1997.
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Livingston, Schuyler, Benjamin Young, Martin Markowitz, Poonam Mathur i Bruce L. Gilliam. HIV Virology. Oxford University Press, 2017. http://dx.doi.org/10.1093/med/9780190493097.003.0017.
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HIV is a member of the lentivirus subfamily of retroviruses. Two distinct groups of viruses are pathogenic in humans: HIV-1 and HIV-2. Both are transmitted sexually and known to cause immunodeficiency disease. HIV enters the cell through use of the CD4 receptor and chemokine co-receptors, primarily CCR5 and CXCR4. The viral genome is transcribed from RNA to DNA by reverse transcriptase and integrated into the host genome by integrase. The HIV genome encodes 15 proteins, comprising three categories: structural, regulatory, and accessory. After budding from the host cell, the virus matures into its infectious form through cleavage of viral precursor proteins by protease.
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Spearman, Paul, i Eric O. Freed. HIV Interactions with Host Cell Proteins. Springer Berlin / Heidelberg, 2012.
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Spearman, Paul, i Eric O. Freed. HIV Interactions with Host Cell Proteins. Springer, 2010.
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Enterotoxins: Microbial Proteins and Host Cell Dysregulation. MDPI, 2016. http://dx.doi.org/10.3390/books978-3-03842-164-1.
Pełny tekst źródłaCzęści książek na temat "CHO HOST CELL PROTEINS"
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Hogwood, Catherine E. M., Lesley M. Chiverton i C. Mark Smales. "Characterization of Host Cell Proteins (HCPs) in CHO Cell Bioprocesses". W Methods in Molecular Biology, 243–50. New York, NY: Springer New York, 2017. http://dx.doi.org/10.1007/978-1-4939-6972-2_16.
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Rasmussen, Brian, Ray Davis, James Thomas i Pranhitha Reddy. "Isolation, characterization and recombinant protein expression in Veggie-CHO: A serum-free CHO host cell line". W Current Applications of Cell Culture Engineering, 31–42. Dordrecht: Springer Netherlands, 1998. http://dx.doi.org/10.1007/978-94-011-4786-6_4.
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Zhang, Peiqing, Kah Fai Chan, Ryan Haryadi, Muriel Bardor i Zhiwei Song. "CHO Glycosylation Mutants as Potential Host Cells to Produce Therapeutic Proteins with Enhanced Efficacy". W Advances in Biochemical Engineering/Biotechnology, 63–87. Berlin, Heidelberg: Springer Berlin Heidelberg, 2012. http://dx.doi.org/10.1007/10_2012_163.
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Kunert, R., K. Strutzenberger, F. Steindl, A. Zudjelovic, N. Borth i H. Katinger. "Protein Mass Production in Hybridomas and Recombinant CHO Cells". W Recombinant Protein Production with Prokaryotic and Eukaryotic Cells. A Comparative View on Host Physiology, 89–97. Dordrecht: Springer Netherlands, 2001. http://dx.doi.org/10.1007/978-94-015-9749-4_7.
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Bocking, Sharon P., Sarah E. Steane, Sureeta Saini i Alan D. Bennett. "Optimizing the Production of Recombinant Prion Protein from CHO Cells". W Recombinant Protein Production with Prokaryotic and Eukaryotic Cells. A Comparative View on Host Physiology, 319–29. Dordrecht: Springer Netherlands, 2001. http://dx.doi.org/10.1007/978-94-015-9749-4_23.
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Schiesser, William E. "Host Cell Proteins with Cross Diffusion". W Virus Host Cell Genetic Material Transport, 35–60. Cham: Springer International Publishing, 2021. http://dx.doi.org/10.1007/978-3-030-68865-3_3.
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Honda, Tomoyuki, i Keizo Tomonaga. "Host Molecular Chaperones: Cell Surface Receptors for Viruses". W Heat Shock Proteins, 293–307. Dordrecht: Springer Netherlands, 2013. http://dx.doi.org/10.1007/978-94-007-6787-4_19.
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Teter, Ken. "Cholera Toxin Interactions with Host Cell Stress Proteins". W Heat Shock Proteins, 323–38. Dordrecht: Springer Netherlands, 2013. http://dx.doi.org/10.1007/978-94-007-6787-4_21.
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Berry, A. M., U. Rasmussen, K. Bateman, S. Lindwall, K. Huss-Danell i B. Bergman. "Arabinogalactan-Protein Epitopes Are Host-Derived in Frankia-Alnus Symbiosis". W Cell and Developmental Biology of Arabinogalactan-Proteins, 281–82. Boston, MA: Springer US, 2000. http://dx.doi.org/10.1007/978-1-4615-4207-0_28.
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Doneanu, Catalin E., i Weibin Chen. "Analysis of Host-Cell Proteins in Biotherapeutic Proteins by LC/MS Approaches". W Methods in Molecular Biology, 341–50. Totowa, NJ: Humana Press, 2014. http://dx.doi.org/10.1007/978-1-62703-977-2_25.
Pełny tekst źródłaStreszczenia konferencji na temat "CHO HOST CELL PROTEINS"
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Wang, Jianye, Kaixuan Zhu, Gang Zhao i Dayong Gao. "Effect of Temperature and Cryoprotectant Solutes on Water Permeability of SF21 Cell Membrane". W ASME 2013 Summer Bioengineering Conference. American Society of Mechanical Engineers, 2013. http://dx.doi.org/10.1115/sbc2013-14056.
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Insect cell as a host of viruses is extensively used for producing heterologous recombinant proteins. The eukaryotic proteins expressed by the insect cell is posttranslationally modified and harvested in a short period of time. The insect cell expression has been applied to both basic research and commercial applications. A large scale of proteins produced by the insect cell expression system are settled for researching the structure and function of the eukaryotic proteins, and the expression system integrated with routine biochemical techniques plays a significant role in diagnostic procedure and therapeutic approaches for the disease.
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Small, Gerald J. "Hole-Burning in Biological Systems". W Spectral Hole-Burning and Luminescence Line Narrowing: Science and Applications. Washington, D.C.: Optica Publishing Group, 1992. http://dx.doi.org/10.1364/shbl.1992.tua2.
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Understanding the excited state electronic structure, optical excitation (energy) transfer and primary charge separation in photosynthetic protein-chlorophyll complexes represents an important but formidable problem [1]. At the outset it is important to realize that the protein, as a host for the various pigments (cofactors), is glass-like, meaning that the optical absorption bands of pigments such as chlorophylls (Chl) suffer from significant site inhomogeneous broadening (ΓI). For Chl and pheophytin cofactors the absorption band of primary interest is the origin or (0,0) band of the lowest excited singlet state which is commonly referred to as the Qy-state, a ππ* state. Such bands carry widths from ~ 100-500 cm-1, depending on the system. For several reasons it becomes important to unravel the ΓI and homogeneous broadening (ΓH) contributions to the bandwidth. Two important contributors to ΓH are the linear electron-phonon coupling (Γep) and exciton level structure accompanied by ultra-fast inter-exciton level relaxation (Γex). It turns out that Γex is most important for antenna protein complexes because their unit cells (of a 2-dimensional array) often contain several strongly interacting Chls.
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Limper, Andrew H., i Theodore J. Kottom. "The Pneumocystis Carinii PcAce2 Transcription Factor Regulates Cell Wall Remodeling Induced By Organism Adhesion To Host Matrix Proteins". W American Thoracic Society 2012 International Conference, May 18-23, 2012 • San Francisco, California. American Thoracic Society, 2012. http://dx.doi.org/10.1164/ajrccm-conference.2012.185.1_meetingabstracts.a3297.
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Khan, Mohammed, Xiaolin Chen i Jie Xu. "Separation of Non-Viable Chinese Hamster Ovary (CHO) Cells Using Dielectrophoresis in a Deterministic Lateral Displacement Ratchet". W ASME 2020 International Mechanical Engineering Congress and Exposition. American Society of Mechanical Engineers, 2020. http://dx.doi.org/10.1115/imece2020-23520.
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Abstract Chinese hamster ovary (CHO) cell is the most widely used mammalian cell line for commercial production of therapeutic protein. Any presence of non-viable cells in culture medium may adversely affect subsequent functionality of these proteins. Therefore, separation of non-viable cells from suspending medium is critical in biopharmaceutical and biomedical sectors. One such method termed Deterministic Lateral Displacement has already shown promising capabilities in separating cells based on the cell size difference by taking advantage of the predictable flow laminae. However, in cases where size overlaps between viable and non-viable cells are present, separation based solely on size suffers and high-resolution separation techniques are required. Dielectrophoresis, one of the most widely used nonlinear electro-kinetic mechanism, has the potential to manipulate the same size cells depending on the dielectric properties of individual cells. In this work, we demonstrated that a DLD device can be combined with a frequency-based AC electric field to perform high resolution continuous separation of non-viable CHO cells from the viable or productive cells. The behavior of the coupled DLD-DEP device is further investigated by employing numerical simulation to check the effect of geometrical parameters of the DLD arrays, velocities of the flow field and required applied voltages. A moderate row shift fraction with velocity 700μm/s provided a good separation behavior without any trapping. The cell viability was also ensured by maintaining proper electric field which otherwise may cause cell loss due to ion leakage. Our developed numerical model and presented results lay the groundwork for design and fabrication of high resolution DLD-DEP microchips for enhanced separation of viable and nonviable cells.
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Patwardhan, Anshuman V., Mohammad M. Ataai i Mansour Zenouzi. "Mathematical Modeling and Optimization of the Immobilized Metal Affinity Chromatography". W ASME 1996 International Mechanical Engineering Congress and Exposition. American Society of Mechanical Engineers, 1996. http://dx.doi.org/10.1115/imece1996-1195.
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Abstract Immobilized copper affinity chromatography experiments with E. coli cell extract have demonstrated that the cellular proteins of this most commonly used host organism elute from the column in a discontinuous fashion characterized by two major peaks. This result has interesting implications towards the mathematical modeling and subsequent optimization of IMAC. Namely, the elution behavior of the E. coli cell extract can be approximately simulated by simply two representative proteins that have a similar elution behavior as that of the peaks. We have identified the mixture of Bovine Serum Albumin and Tuna Heart Cytochrome-C to be excellent representatives of the E. coli cell extract. The equilibrium and transport parameters of these proteins were determined experimentally and used in a detailed mathematical model incorporating multicomponent Langmuir isotherm, axial dispersion, film mass transfer and intraparticle diffusion. Target proteins of varying affinities were also included in the model. Simulation results provided an excellent framework for identifying effective strategies for optimal column utilization as well as for attaining high resolution of the target protein.
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Zhang, Jun, Sheng Yan, Dan Yuan, Gursel Alici, Nam-trung Nguyen i Weihua Li. "High Throughput Cell-Free Extraction of Plasma by an Integrated Microfluidic Device Combining Inertial Microfluidics and Membrane". W ASME 2016 5th International Conference on Micro/Nanoscale Heat and Mass Transfer. American Society of Mechanical Engineers, 2016. http://dx.doi.org/10.1115/mnhmt2016-6717.
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Plasma is a host of various analytes such as proteins, metabolites, circulating nucleic acids (CNAs), pathogens. The key process of plasma extraction is to eliminate the contamination from blood cells. Conventional methods, such as centrifugation and membrane filtration, are generally lab-intensive, time consuming and even dangerous. In this study, we report an integrated microfluidic device that combines inertial microfluidics and membrane filter. The integrated microfluidic device was evaluated by the diluted (x1/10, x1/20) whole blood, and the quality of the extracted blood plasma was tested. It was found that quality of extracted blood plasma from integrated device was equivalent to that obtained by the centrifugation. This study demonstrates a significant progress towards the practical application of inertial microfluidics with membrane filter for high-throughput and high efficient blood plasma extraction.
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Tan Hui En, Glenda, Koay Tze Erhn i Shen Bingquan. "Anti-Virus Autobots: Predicting More Infectious Virus Variants for Pandemic Prevention through Deep Learning". W 4th International Conference on Machine Learning & Applications (CMLA 2022). Academy and Industry Research Collaboration Center (AIRCC), 2022. http://dx.doi.org/10.5121/csit.2022.121102.
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More infectious virus variants can arise from rapid mutations in their proteins, creating new infection waves. These variants can evade one’s immune system and infect vaccinated individuals, lowering vaccine efficacy. Hence, to improve vaccine design, this project proposes Optimus PPIme – a deep learning approach to predict future, more infectious variants from an existing virus (exemplified by SARS-CoV-2). The approach comprises an algorithm which acts as a “virus” attacking a host cell. To increase infectivity, the “virus” mutates to bind better to the host’s receptor. 2 algorithms were attempted – greedy search and beam search. The strength of this variant-host binding was then assessed by a transformer network we developed, with a high accuracy of 90%. With both components, beam search eventually proposed more infectious variants. Therefore, this approach can potentially enable researchers to develop vaccines that provide protection against future infectious variants before they emerge, preempting outbreaks and saving lives.
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ZOMER, G., M. HAMZINK, A. DE HAAN, G. KERSTEN i K. REUBSAET. "DEVELOPMENT AND OPTIMIZATION OF A QUANTITATIVE WESTERN BLOT AND DOT BLOT PROCEDURE FOR THE DETERMINATION OF RESIDUAL HOST CELL PROTEINS PRESENT IN INACTIVATED POLIO VACCINE USING A GZ11 BASED SIGNAL REAGENT". W Proceedings of the 15th International Symposium. WORLD SCIENTIFIC, 2008. http://dx.doi.org/10.1142/9789812839589_0066.
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Karges, H. E., G. Zettlemeiβl, H. Naumann, U. Eberhard i M. Bröker. "PURIFICATION AND CHARACTERIZATION OF GENTECHNOLOGICALLY PREPARED ANTITHROMBIN III". W XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643684.
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Isolation and purification of antithrombin III (AT III) by affinity chromatography on immobilized heparin is a standard method for the large scale preparation of this protein from human or animal plasma. Hence, after AT III became available by gentechnological methods, we tried to adapt this procedure for the isolation of AT III from supernatants of mammalian- and yeast-cells. Indeed, it was possible to use this method also for the isolation of the recombinant gene products. Since, however, the cell growth media contain heterologous protein or peptide mixtures like fetal calf serum, the method had to be improved to avoid the adsorption of non human proteins or peptides. We are now able to purify AT III from CHO-cell-superna-tants to more than 95 % purity. The characterization of this AT III-product by double immuno diffusion revealed that it is immunologically totally identical with the authentic material from plasma. AT III antigen content, progressive inhibitor activity and heparin cofactor activity compare very well in the final product; hence, it is totally active compared to AT III from plasma.In polyacrylamidegel electrophoresis most of the material migrated differently to the authentic material showing 9 bands in equal distance to each other, instead four in the At III from plasma. After degradation with sialinidase from both AT III preparations identical cleavage products were obtained migrating predominantly as a single band. Hence, the electrophoretic heterogeneity seems to be due to a different degree of sialinyla-tion of the products.
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Rickles, F. R., W. W. Hancock, K. Kobzik, N. Hogg i C. O’Hara. "THE DISTRIBUTION OF CROSS-LINKED FIBRIN IN HUMAN LUNG CARCINOMA PARALLELS THAT OF ACTIVATED HOST MONONUCLEAR LEUKOCYTES: IMMUNO-HISTOLOGIC STUDIES WITH MONOCLONAL ANTIBODIES". W XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643669.
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Previous immunohistologic studies of human lung carcinoma, using polyclonal antibodies to antigens shared between fibrinogen (FGN) and fibrin (FB), showed that FGN/FB were associated withtumor cells. These findings were interpreted as evidence of the presence of tumor-associated procoagulant activity(PCA). Wecompared the distribution of coagulation-associated proteins in 16 casesof human carcinoma of the lungofvarying histologic types, using polyclonal antibodies to FGN/FB and monoclonal antibodies (mAb) to cross-linked fibrin (XL-FB;UC-45), fibronectin (FN;PHM13), factor VIII (vWF:Ag)and a tissue factor-related antigen(TF:RAg;A1 -3)- Host mononuclear leukocytes were identifiedusing various mAb toT cells and macrophages, and studied for their expression of receptorsfor interleukin-2 (IL-2R). Positive resultsare summarizedIn addition, studies of the mononuclearcells adjacent to tumors in 12/12 casesshowed the presence of tumor-associated macrophages, 10/11 showed T cells,mainly T8+, and A/5 showed corresponding expression of IL-2R, suggesting cell activation.The use of highly specific mAb showed that XL-FB is actually more selectively distributed than is found using polyclonal antisera to FGN/FB, and indeed XL-FB was largely confined to those areas adjacent to tumors which are rich in mononuclearcells. These findings suggest that fibrin deposits in human carcinomasof the lung may be due todevelopment of PCA by activated host mononuclear cells, rather than tumor cells.This lack of XL-FB on tumor cellsinspite of A1- 3 binding suggests that TF:RAg may not be available on the tumor cell surface for the activation of clotting. Further studies are neededto define the functional capacity of PCA molecules on tumor cells and tumor-associated mononuclear cells in situ.
Raporty organizacyjne na temat "CHO HOST CELL PROTEINS"
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Epel, Bernard, i Roger Beachy. Mechanisms of intra- and intercellular targeting and movement of tobacco mosaic virus. United States Department of Agriculture, listopad 2005. http://dx.doi.org/10.32747/2005.7695874.bard.
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To cause disease, plant viruses must replicate and spread locally and systemically within the host. Cell-to-cell virus spread is mediated by virus-encoded movement proteins (MPs), which modify the structure and function of plasmodesmata (Pd), trans-wall co-axial membranous tunnels that interconnect the cytoplasm of neighboring cells. Tobacco mosaic virus (TMV) employ a single MP for cell- cell spread and for which CP is not required. The PIs, Beachy (USA) and Epel (Israel) and co-workers, developed new tools and approaches for study of the mechanism of spread of TMV that lead to a partial identification and molecular characterization of the cellular machinery involved in the trafficking process. Original research objectives: Based on our data and those of others, we proposed a working model of plant viral spread. Our model stated that MPᵀᴹⱽ, an integral ER membrane protein with its C-terminus exposed to the cytoplasm (Reichel and Beachy, 1998), alters the Pd SEL, causes the Pd cytoplasmic annulus to dilate (Wolf et al., 1989), allowing ER to glide through Pd and that this gliding is cytoskeleton mediated. The model claimed that in absence of MP, the ER in Pd (the desmotubule) is stationary, i.e. does not move through the Pd. Based on this model we designed a series of experiments to test the following questions: -Does MP potentiate ER movement through the Pd? - In the presence of MP, is there communication between adjacent cells via ER lumen? -Does MP potentiate the movement of cytoskeletal elements cell to cell? -Is MP required for cell-to-cell movement of ER membranes between cells in sink tissue? -Is the binding in situ of MP to RNA specific to vRNA sequences or is it nonspecific as measured in vitro? And if specific: -What sequences of RNA are involved in binding to MP? And finally, what host proteins are associated with MP during intracellular targeting to various subcellular targets and what if any post-translational modifications occur to MP, other than phosphorylation (Kawakami et al., 1999)? Major conclusions, solutions and achievements. A new quantitative tool was developed to measure the "coefficient of conductivity" of Pd to cytoplasmic soluble proteins. Employing this tool, we measured changes in Pd conductivity in epidermal cells of sink and source leaves of wild-type and transgenic Nicotiana benthamiana (N. benthamiana) plants expressing MPᵀᴹⱽ incubated both in dark and light and at 16 and 25 ᵒC (Liarzi and Epel, 2005 (appendix 1). To test our model we measured the effect of the presence of MP on cell-to-cell spread of a cytoplasmic fluorescent probe, of two ER intrinsic membrane protein-probes and two ER lumen protein-probes fused to GFP. The effect of a mutant virus that is incapable of cell-to-cell spread on the spread of these probes was also determined. Our data shows that MP reduces SEL for cytoplasmic molecules, dilates the desmotubule allowing cell-cell diffusion of proteins via the desmotubule lumen and reduces the rate of spread of the ER membrane probes. Replicase was shown to enhance cell-cell spread. The data are not in support of the proposed model and have led us to propose a new model for virus cell-cell spread: this model proposes that MP, an integral ER membrane protein, forms a MP:vRNAER complex and that this ER-membrane complex diffuses in the lipid milieu of the ER into the desmotubule (the ER within the Pd), and spreads cell to cell by simple diffusion in the ER/desmotubule membrane; the driving force for spread is the chemical potential gradient between an infected cell and contingent non-infected neighbors. Our data also suggests that the virus replicase has a function in altering the Pd conductivity. Transgenic plant lines that express the MP gene of the Cg tobamovirus fused to YFP under the control the ecdysone receptor and methoxyfenocide ligand were generated by the Beachy group and the expression pattern and the timing and targeting patterns were determined. A vector expressing this MPs was also developed for use by the Epel lab . The transgenic lines are being used to identify and isolate host genes that are required for cell-to-cell movement of TMV/tobamoviruses. This line is now being grown and to be employed in proteomic studies which will commence November 2005. T-DNA insertion mutagenesis is being developed to identify and isolate host genes required for cell-to-cell movement of TMV.
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Epel, Bernard L., Roger N. Beachy, A. Katz, G. Kotlinzky, M. Erlanger, A. Yahalom, M. Erlanger i J. Szecsi. Isolation and Characterization of Plasmodesmata Components by Association with Tobacco Mosaic Virus Movement Proteins Fused with the Green Fluorescent Protein from Aequorea victoria. United States Department of Agriculture, wrzesień 1999. http://dx.doi.org/10.32747/1999.7573996.bard.
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The coordination and regulation of growth and development in multicellular organisms is dependent, in part, on the controlled short and long-distance transport of signaling molecule: In plants, symplastic communication is provided by trans-wall co-axial membranous tunnels termed plasmodesmata (Pd). Plant viruses spread cell-to-cell by altering Pd. This movement scenario necessitates a targeting mechanism that delivers the virus to a Pd and a transport mechanism to move the virion or viral nucleic acid through the Pd channel. The identity of host proteins with which MP interacts, the mechanism of the targeting of the MP to the Pd and biochemical information on how Pd are alter are questions which have been dealt with during this BARD project. The research objectives of the two labs were to continue their biochemical, cellular and molecular studies of Pd composition and function by employing infectious modified clones of TMV in which MP is fused with GFP. We examined Pd composition, and studied the intra- and intercellular targeting mechanism of MP during the infection cycle. Most of the goals we set for ourselves were met. The Israeli PI and collaborators (Oparka et al., 1999) demonstrated that Pd permeability is under developmental control, that Pd in sink tissues indiscriminately traffic proteins of sizes of up to 50 kDa and that during the sink to source transition there is a substantial decrease in Pd permeability. It was shown that companion cells in source phloem tissue export proteins which traffic in phloem and which unload in sink tissue and move cell to cell. The TAU group employing MP:GFP as a fluorescence probe for optimized the procedure for Pd isolation. At least two proteins kinases found to be associated with Pd isolated from source leaves of N. benthamiana, one being a calcium dependent protein kinase. A number of proteins were microsequenced and identified. Polyclonal antibodies were generated against proteins in a purified Pd fraction. A T-7 phage display library was created and used to "biopan" for Pd genes using these antibodies. Selected isolates are being sequenced. The TAU group also examined whether the subcellular targeting of MP:GFP was dependent on processes that occurred only in the presence of the virus or whether targeting was a property indigenous to MP. Mutant non-functional movement proteins were also employed to study partial reactions. Subcellular targeting and movement were shown to be properties indigenous to MP and that these processes do not require other viral elements. The data also suggest post-translational modification of MP is required before the MP can move cell to cell. The USA group monitored the development of the infection and local movement of TMV in N. benthamiana, using viral constructs expressing GFP either fused to the MP of TMV or expressing GFP as a free protein. The fusion protein and/or the free GFP were expressed from either the movement protein subgenomic promoter or from the subgenomic promoter of the coat protein. Observations supported the hypothesis that expression from the cp sgp is regulated differently than expression from the mp sgp (Szecsi et al., 1999). Using immunocytochemistry and electron microscopy, it was determined that paired wall-appressed bodies behind the leading edge of the fluorescent ring induced by TMV-(mp)-MP:GFP contain MP:GFP and the viral replicase. These data suggest that viral spread may be a consequence of the replication process. Observation point out that expression of proteins from the mp sgp is temporary regulated, and degradation of the proteins occurs rapidly or more slowly, depending on protein stability. It is suggested that the MP contains an external degradation signal that contributes to rapid degradation of the protein even if expressed from the constitutive cp sgp. Experiments conducted to determine whether the degradation of GFP and MP:GFP was regulated at the protein or RNA level, indicated that regulation was at the protein level. RNA accumulation in infected protoplast was not always in correlation with protein accumulation, indicating that other mechanisms together with RNA production determine the final intensity and stability of the fluorescent proteins.
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Manulis, Shulamit, Christine D. Smart, Isaac Barash, Guido Sessa i Harvey C. Hoch. Molecular Interactions of Clavibacter michiganensis subsp. michiganensis with Tomato. United States Department of Agriculture, styczeń 2011. http://dx.doi.org/10.32747/2011.7697113.bard.
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Clavibacter michiganensis subsp. michiganensis (Cmm), the causal agent of bacterial wilt and canker of tomato, is the most destructive bacterial disease of tomato causing substantial economic losses in Israel, the U.S.A. and worldwide. The molecular strategies that allow Cmm, a Gram-positive bacterium, to develop a successful infection in tomato plants are largely unknown. The goal of the project was to elucidate the molecular interactions between Cmmand tomato. The first objective was to analyze gene expression profiles of susceptible tomato plants infected with pathogenic and endophytic Cmmstrains. Microarray analysis identified 122 genes that were differentially expressed during early stages of infection. Cmm activated typical basal defense responses in the host including induction of defense-related genes, production of scavenging of free oxygen radicals, enhanced protein turnover and hormone synthesis. Proteomic investigation of the Cmm-tomato interaction was performed with Multi-Dimensional Protein Identification Technology (MudPIT) and mass spectroscopy. A wide range of enzymes secreted by Cmm382, including cell-wall degrading enzymes and a large group of serine proteases from different families were identified in the xylem sap of infected tomato. Based on proteomic results, the expression pattern of selected bacterial virulence genes and plant defense genes were examined by qRT-PCR. Expression of the plasmid-borne cellulase (celA), serine protease (pat-1) and serine proteases residing on the chp/tomA pathogenicity island (chpCandppaA), were significantly induced within 96 hr after inoculation. Transcription of chromosomal genes involved in cell wall degradation (i.e., pelA1, celB, xysA and xysB) was also induced in early infection stages. The second objective was to identify by VIGS technology host genes affecting Cmm multiplication and appearance of disease symptoms in plant. VIGS screening showed that out of 160 tomato genes, which could be involved in defense-related signaling, suppression of 14 genes led to increase host susceptibility. Noteworthy are the genes Snakin-2 (inhibitor of Cmm growth) and extensin-like protein (ELP) involved in cell wall fortification. To further test the significance of Snakin -2 and ELP in resistance towards Cmm, transgenic tomato plants over-expressing the two genes were generated. These plants showed partial resistance to Cmm resulting in a significant delay of the wilt symptoms and reduction in size of canker lesion compared to control. Furthermore, colonization of the transgenic plants was significantly lower. The third objective was to assess the involvement of ethylene (ET), jasmonate (JA) and salicylic acid (SA) in Cmm infection. Microarray and proteomic studies showed the induction of enzymes involved in ET and JA biosynthesis. Cmm promoted ET production 8 days after inoculation and SIACO, a key enzyme of ET biosynthesis, was upregulated. Inoculation of the tomato mutants Never ripe (Nr) impaired in ET perception and transgenic plants with reduced ET synthesis significantly delayed wilt symptoms as compared to the wild-type plants. The retarded wilting in Nr plants was shown to be a specific effect of ET insensitivity and was not due to altered expression of defense related genes, reduced bacterial population or decrease in ethylene biosynthesis . In contrast, infection of various tomato mutants impaired in JA biosynthesis (e.g., def1, acx1) and JA insensitive mutant (jai1) yielded unequivocal results. The fourth objective was to determine the role of cell wall degrading enzymes produced by Cmm in xylem colonization and symptoms development. A significance increase (2 to 7 fold) in expression of cellulases (CelA, CelB), pectate lyases (PelA1, PelA2), polygalacturonase and xylanases (XylA, XylB) was detected by qRT-PCR and by proteomic analysis of the xylem sap. However, with the exception of CelA, whose inactivation led to reduced wilt symptoms, inactivation of any of the other cell wall degrading enzymes did not lead to reduced virulence. Results achieved emphasized the complexity involved in Cmm-tomato interactions. Nevertheless they provide the basis for additional research which will unravel the mechanism of Cmm pathogenicity and formulating disease control measures.
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Droby, Samir, Michael Wisniewski, Ron Porat i Dumitru Macarisin. Role of Reactive Oxygen Species (ROS) in Tritrophic Interactions in Postharvest Biocontrol Systems. United States Department of Agriculture, grudzień 2012. http://dx.doi.org/10.32747/2012.7594390.bard.
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To elucidate the role of ROS in the tri-trophic interactions in postharvest biocontrol systems a detailed molecular and biochemical investigation was undertaken. The application of the yeast biocontrol agent Metschnikowia fructicola, microarray analysis was performed on grapefruit surface wounds using an Affymetrix Citrus GeneChip. the data indicated that 1007 putative unigenes showed significant expression changes following wounding and yeast application relative to wounded controls. The expression of the genes encoding Respiratory burst oxidase (Rbo), mitogen-activated protein kinase (MAPK) and mitogen-activated protein kinase kinase (MAPKK), G-proteins, chitinase (CHI), phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS) and 4-coumarate-CoA ligase (4CL). In contrast, three genes, peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT), were down-regulated in grapefruit peel tissue treated with yeast cells. The yeast antagonists, Metschnikowia fructicola (strain 277) and Candida oleophila (strain 182) generate relatively high levels of super oxide anion (O2−) following its interaction with wounded fruit surface. Using laser scanning confocal microscopy we observed that the application of M. fructicola and C. oleophila into citrus and apple fruit wounds correlated with an increase in H2O2 accumulation in host tissue. The present data, together with our earlier discovery of the importance of H₂O₂ production in the defense response of citrus flavedo to postharvest pathogens, indicate that the yeast-induced oxidative response in fruit exocarp may be associated with the ability of specific yeast species to serve as biocontrol agents for the management of postharvest diseases. Effect of ROS on yeast cells was also studied. Pretreatment of the yeast, Candida oleophila, with 5 mM H₂O₂ for 30 min (sublethal) increased yeast tolerance to subsequent lethal levels of oxidative stress (50 mM H₂O₂), high temperature (40 °C), and low pH (pH 4). Suppression subtractive hybridization analysis was used to identify genes expressed in yeast in response to sublethal oxidative stress. Transcript levels were confirmed using semi quantitative reverse transcription-PCR. Seven antioxidant genes were up regulated. Pretreatment of the yeast antagonist Candida oleophila with glycine betaine (GB) increases oxidative stress tolerance in the microenvironment of apple wounds. ROS production is greater when yeast antagonists used as biocontrol agents are applied in the wounds. Compared to untreated control yeast cells, GB-treated cells recovered from the oxidative stress environment of apple wounds exhibited less accumulation of ROS and lower levels of oxidative damage to cellular proteins and lipids. Additionally, GB-treated yeast exhibited greater biocontrol activity against Penicillium expansum and Botrytis cinerea, and faster growth in wounds of apple fruits compared to untreated yeast. The expression of major antioxidant genes, including peroxisomal catalase, peroxiredoxin TSA1, and glutathione peroxidase was elevated in the yeast by GB treatment. A mild heat shock (HS) pretreatment (30 min at 40 1C) improved the tolerance of M. fructicola to subsequent high temperature (45 1C, 20–30 min) and oxidative stress (0.4 mol-¹) hydrogen peroxide, 20–60 min). HS-treated yeast cells showed less accumulation of reactive oxygen species (ROS) than non-treated cells in response to both stresses. Additionally, HS-treated yeast exhibited significantly greater (P≥0.0001) biocontrol activity against Penicillium expansum and a significantly faster (Po0.0001) growth rate in wounds of apple fruits stored at 25 1C compared with the performance of untreated yeast cells. Transcription of a trehalose-6-phosphate synthase gene (TPS1) was up regulated in response to HS and trehalose content also increased.
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Tzfira, Tzvi, Michael Elbaum i Sharon Wolf. DNA transfer by Agrobacterium: a cooperative interaction of ssDNA, virulence proteins, and plant host factors. United States Department of Agriculture, grudzień 2005. http://dx.doi.org/10.32747/2005.7695881.bard.
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Agrobacteriumtumefaciensmediates genetic transformation of plants. The possibility of exchanging the natural genes for other DNA has led to Agrobacterium’s emergence as the primary vector for genetic modification of plants. The similarity among eukaryotic mechanisms of nuclear import also suggests use of its active elements as media for non-viral genetic therapy in animals. These considerations motivate the present study of the process that carries DNA of bacterial origin into the host nucleus. The infective pathway of Agrobacterium involves excision of a single-stranded DNA molecule (T-strand) from the bacterial tumor-inducing plasmid. This transferred DNA (T-DNA) travels to the host cell cytoplasm along with two virulence proteins, VirD2 and VirE2, through a specific bacteriumplant channel(s). Little is known about the precise structure and composition of the resulting complex within the host cell and even less is known about the mechanism of its nuclear import and integration into the host cell genome. In the present proposal we combined the expertise of the US and Israeli labs and revealed many of the biophysical and biological properties of the genetic transformation process, thus enhancing our understanding of the processes leading to nuclear import and integration of the Agrobacterium T-DNA. Specifically, we sought to: I. Elucidate the interaction of the T-strand with its chaperones. II. Analyzing the three-dimensional structure of the T-complex and its chaperones in vitro. III. Analyze kinetics of T-complex formation and T-complex nuclear import. During the past three years we accomplished our goals and made the following major discoveries: (1) Resolved the VirE2-ssDNA three-dimensional structure. (2) Characterized VirE2-ssDNA assembly and aggregation, along with regulation by VirE1. (3) Studied VirE2-ssDNA nuclear import by electron tomography. (4) Showed that T-DNA integrates via double-stranded (ds) intermediates. (5) Identified that Arabidopsis Ku80 interacts with dsT-DNA intermediates and is essential for T-DNA integration. (6) Found a role of targeted proteolysis in T-DNA uncoating. Our research provide significant physical, molecular, and structural insights into the Tcomplex structure and composition, the effect of host receptors on its nuclear import, the mechanism of T-DNA nuclear import, proteolysis and integration in host cells. Understanding the mechanical and molecular basis for T-DNA nuclear import and integration is an essential key for the development of new strategies for genetic transformation of recalcitrant plant species. Thus, the knowledge gained in this study can potentially be applied to enhance the transformation process by interfering with key steps of the transformation process (i.e. nuclear import, proteolysis and integration). Finally, in addition to the study of Agrobacterium-host interaction, our research also revealed some fundamental insights into basic cellular mechanisms of nuclear import, targeted proteolysis, protein-DNA interactions and DNA repair.
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Coplin, David L., Shulamit Manulis i Isaac Barash. roles Hrp-dependent effector proteins and hrp gene regulation as determinants of virulence and host-specificity in Erwinia stewartii and E. herbicola pvs. gypsophilae and betae. United States Department of Agriculture, czerwiec 2005. http://dx.doi.org/10.32747/2005.7587216.bard.
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Gram-negative plant pathogenic bacteria employ specialized type-III secretion systems (TTSS) to deliver an arsenal of pathogenicity proteins directly into host cells. These secretion systems are encoded by hrp genes (for hypersensitive response and pathogenicity) and the effector proteins by so-called dsp or avr genes. The functions of effectors are to enable bacterial multiplication by damaging host cells and/or by blocking host defenses. We characterized essential hrp gene clusters in the Stewart's Wilt of maize pathogen, Pantoea stewartii subsp. stewartii (Pnss; formerly Erwinia stewartii) and the gall-forming bacterium, Pantoea agglomerans (formerly Erwinia herbicola) pvs. gypsophilae (Pag) and betae (Pab). We proposed that the virulence and host specificity of these pathogens is a function of a) the perception of specific host signals resulting in bacterial hrp gene expression and b) the action of specialized signal proteins (i.e. Hrp effectors) delivered into the plant cell. The specific objectives of the proposal were: 1) How is the expression of the hrp and effector genes regulated in response to host cell contact and the apoplastic environment? 2) What additional effector proteins are involved in pathogenicity? 3) Do the presently known Pantoea effector proteins enter host cells? 4) What host proteins interact with these effectors? We characterized the components of the hrp regulatory cascade (HrpXY ->7 HrpS ->7 HrpL ->7 hrp promoters), showed that they are conserved in both Pnss and Fag, and discovered that the regulation of the hrpS promoter (hrpSp) may be a key point in integrating apoplastic signals. We also analyzed the promoters recognized by HrpL and demonstrated the relationship between their composition and efficiency. Moreover, we showed that promoter strength can influence disease expression. In Pnss, we found that the HrpXY two-component signal system may sense the metabolic status of the bacterium and is required for full hrp gene expression in planta. In both species, acyl-homoserine lactone-mediated quorum sensing may also regulate epiphytic fitness and/or pathogenicity. A common Hrp effector protein, DspE/WtsE, is conserved and required for virulence of both species. When introduced into corn cells, Pnss WtsE protein caused water-soaked lesions. In other plants, it either caused cell death or acted as an Avr determinant. Using a yeast- two-hybrid system, WtsE was shown to interact with a number of maize signal transduction proteins that are likely to have roles in either programmed cell death or disease resistance. In Pag and Pab, we have characterized the effector proteins HsvG, HsvB and PthG. HsvG and HsvB are homologous proteins that determine host specificity of Pag and Pab on gypsophila and beet, respectively. Both possess a transcriptional activation domain that functions in yeast. PthG was found to act as an Avr determinant on multiple beet species, but was required for virulence on gypsophila. In addition, we demonstrated that PthG acts within the host cell. Additional effector genes have been characterized on the pathogenicity plasmid, pPATHₚₐg, in Pag. A screen for HrpL- regulated genes in Pnsspointed up 18 candidate effector proteins and four of these were required for full virulence. It is now well established that the virulence of Gram-negative plant pathogenic bacteria is governed by Hrp-dependent effector proteins. However; the mode of action of many effectors is still unresolved. This BARD supported research will significantly contribute to the understanding of how Hrp effectors operate in Pantoea spp. and how they control host specificity and affect symptom production. This may lead to novel approaches for genetically engineering plants resistant to a wide range of bacterial pathogens by inactivating the Hrp effectors with "plantabodies" or modifying their receptors, thereby blocking the induction of the susceptible response. Alternatively, innovative technologies could be used to interfere with the Hrp regulatory cascade by blocking a critical step or mimicking plant or quorum sensing signals.
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Banai, Menachem, i Gary Splitter. Molecular Characterization and Function of Brucella Immunodominant Proteins. United States Department of Agriculture, lipiec 1993. http://dx.doi.org/10.32747/1993.7568100.bard.
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The BARD project was a continuation of a previous BARD funded research project. It was aimed at characterization of the 12kDa immunodominant protein and subsequently the cloning and expression of the gene in E. coli. Additional immunodominant proteins were sought among genomic B. abortus expression library clones using T-lymphocyte proliferation assay as a screening method. The 12kDa protein was identified as the L7/L12 ribosomal protein demonstrating in the first time the role a structural protein may play in the development of the host's immunity against the organism. The gene was cloned from B. abortus (USA) and B. melitensis (Israel) showing identity of the oligonucleotide sequence between the two species. Further subcloning allowed expression of the protein in E. coli. While the native protein was shown to have DTH antigenicity its recombinant analog lacked this activity. In contrast the two proteins elicited lymphocyte proliferation in experimental murine brucellosis. CD4+ cells of the Th1 subset predominantly responded to this protein demonstrating the development of protective immunity (g-IFN, and IL-2) in the host. Similar results were obtained with bovine Brucella primed lymphocytes. UvrA, GroE1 and GroEs were additional Brucella immunodominant proteins that demonstrated MHC class II antigenicity. The role cytotoxic cells are playing in the clearance of brucella cells was shown using knock out mice defective either in their CD4+ or CD8+ cells. CD4+ defective mice were able to clear brucella as fast as did normal mice. In contrast mice which were defective in their CD8+ cells could not clear the organisms effectively proving the importance of this subtype cell line in development of protective immunity. The understanding of the host's immune response and the expansion of the panel of Brucella immunodominant proteins opened new avenues in vaccine design. It is now feasible to selectively use immunodominant proteins either as subunit vaccine to fortify immunity of older animals or as diagnostic reagents for the serological survaillance.
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Citovsky, Vitaly, i Yedidya Gafni. Viral and Host Cell Determinants of Nuclear Import and Export of the Tomato Yellow Leaf Curl Virus in Tomato Plants. United States Department of Agriculture, sierpień 2002. http://dx.doi.org/10.32747/2002.7585200.bard.
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Tomato yellow leaf curl geminivirus (TYLCV) is a major pathogen of cultivated tomato, causing up to 100% crop loss in many parts of the world. In Israel, where TYLCV epidemics have been recorded since the 1960' s, this viral disease is well known and has been of economic significance ever since. In recent years, TYLCV outbreaks also occurred in the "New World" - Cuba, The Dominican Republic, and in the USA, in Florida, Georgia and Louisiana. Thus, TYLCV substantially hinders tomato growth throughout the world. Surprisingly, however, little is known about the molecular mechanisms of TYLCV interaction with the host tomato cells. The present proposal, a continuation of the project supported by BARD from 1994, expanded our understanding of the molecular mechanisms by which TYLCV enters the host cell nucleus for replication and transcription and exits it for the subsequent cell-to-cell spread. Our project sought two objectives: I. To study the roles of the viral capsid protein (CP) and host cell factors in TYLCV nuclear import. II. To study the roles of CP and host cell factors in TYLCV nuclear export. Our research toward these goals have produced the following major achievements: . Developed a one-hybrid assay for protein nuclear export and import (#3 in the List of Publications). . Identified a functional nuclear export signal (NES) in the capsid protein (CP) of TYLCV (#3 in the List of Publications). . Discovered homotypic interactions between intact TYLCV CP molecules and analyzed these interactions using deletion mutagenesis of TYLCV CP (#5 in the List of Publications). . Showed developmental and tissue-specific expression of the host factor required for nuclear import of TYLCV CP, tomato karyopherin alpha 1, in transgenic tomato plants (#14 in the List of Publications). . By analogy to nuclear import of TYLCV ,identified an Arabidopsis VIPI protein that participates in nuclear import of Agrobacterium T -complexes via the karyopherin alpha pathway (#4,6, and 8 in the List of Publications). These research findings provided significant insights into (i) the molecular pathway of TYLCV entry into the host cell nucleus, and (ii) the mechanism by which TYLCV is exported from the nucleus for the cell-to-cell spread of infection. Furthermore, the obtained knowledge will help to develop specific strategies to attenuate TYLCV infection, for example, by blocking viral entry into and/or exit out of the host cell nucleus. Also, as much of our findings is relevant to all geminiviruses, new anti- TYLCV approaches developed based on the results of our research will be useful to combat other members of the Geminivirus family. Finally, in addition to the study of TYLCV nuclear import and export, our research contributed to our understanding of general mechanisms for nucleocytoplasmic shuttling of proteins and nucleic acids in plant cells.
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Dickman, Martin B., i Oded Yarden. Characterization of the chorismate mutase effector (SsCm1) from Sclerotinia sclerotiorum. United States Department of Agriculture, styczeń 2015. http://dx.doi.org/10.32747/2015.7600027.bard.
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Sclerotinia sclerotiorum is a filamentous fungus (mold) that causes plant disease. It has an extremely wide range of hosts (>400 species) and causes considerable damage (annual multimillion dollar losses) in economically important crops. It has proven difficult to control (culturally or chemically) and host resistance to this fungus has generally been inadequate. It is believed that this fungus occurs in almost every country. Virulence of this aggressive pathogen is bolstered by a wide array of plant cell wall degrading enzymes and various compounds (secondary metabolites) produced by the fungus. It is well established that plant pathogenic fungi secrete proteins and small molecules that interact with host cells and play a critical role in disease development. Such secreted proteins have been collectively designated as “effectors”. Plant resistance against some pathogens can be mediated by recognition of such effectors. Alternatively, effectors can interfere with plant defense. Some such effectors are recognized by the host plant and can culminate in a programmed cell death (PCD) resistant response. During the course of this study, we analyzed an effector in Sclerotiniasclerotiorum. This specific effector, SsCM1 is the protein chorismatemutase, which is an enzyme involved in a pathway which is important in the production of important amino acids, such a Tryptophan. We have characterized the Sclerotiniaeffector, SsCM1, and have shown that inactivation of Sscm1 does not affect fungal vegetative growth, development or production of oxalic acid (one of this fungus’ secondary metabolites associated with disease) production. However, yhis does result in reduced fungal virulence. We show that, unexpectedly, the SsCM1 protein translocates to the host chloroplast, and demonstrated that this process is required for full fungal virulence. We have also determined that the fungal SsCM1 protein can interact with similar proteins produced by the host. In addition, we have shown that the fungal SsCM1 is able to suppress at least some of the effects imposed by reactive oxygen species which are produced as a defense mechanism by the host. Last, but not least, the results of our studies have provided evidence contradicting the current dogma on at least some of the mechanist aspects of how this pathogen infects the host. Contrary to previousons, indicating that this pathogen kills its host by use of metabolites and enzymes that degrade the host tissue (a process called necrotrophy), we now know that at least in the early phases of infection, the fungus interacts with live host tissue (a phenomenon known as biotrophy). Taken together, the results of our studies provide novel insights concerning the mechanistic aspects of Sclerotinia-host interactions. We hope this information will be used to interfere with the disease cycle in a manner that will protect plants from this devastating fungus.
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Sadot, Einat, Christopher Staiger i Zvi Kam Weizmann. functional genomic screen for new plant cytoskeletal proteins and the determination of their role in actin mediated functions and guard cells regulation. United States Department of Agriculture, styczeń 2003. http://dx.doi.org/10.32747/2003.7587725.bard.
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The original objectives of the approved proposal were: 1. To construct a YFP fused Arabidopsis cDNA library in a mammalian expression vector. 2. To infect the library into a host fibroblast cell line and to screen for new cytoskeletal associated proteins using an automated microscope. 3. Isolate the new genes. 4. Characterize their role in plants. The project was approved as a feasibility study to allow proof of concept that would entail building the YFP library and picking up a couple of positive clones using the fluorescent screen. We report here on the construction of the YFP library, the development of the automatic microscope, the establishment of the screen and the isolation of positive clones that are plant cDNAs encoding cytoskeleton associated proteins. The rational underling a screen of plant library in fibroblasts is based on the high conservation of the cytoskeleton building blocks, actin and tubulin, between the two kingdoms (80-90% homology at the level of amino acids sequence). In addition, several publications demonstrated the recognition of mammalian cytoskeleton by plant cytoskeletal binding proteins and vice versa. The major achievements described here are: 1. The development of an automated microscope equipped with fast laser auto-focusing for high magnification and a software controlling 6 dimensions; X, Y position, auto focus, time, color, and the distribution and density of the fields acquired. This system is essential for the high throughput screen. 2. The construction of an extremely competent YFP library efficiently cloned (tens of thousands of clones collected, no empty vectors detected) with all inserts oriented 5't03'. These parameters render it well representative of the whole transcriptome and efficient in "in-frame" fusion to YFP. 3. The strategy developed for the screen allowing the isolation of individual positive cDNA clones following three rounds of microscopic scans. The major conclusion accomplished from the work described here is that the concept of using mammalian host cells for fishing new plant cytoskeletal proteins is feasible and that screening system developed is complete for addressing one of the major bottlenecks of the plant cytoskeleton field: the need for high throughput identification of functionally active cytoskeletal proteins. The new identified plant cytoskeletal proteins isolated in the pilot screen and additional new proteins which will be isolated in a comprehensive screen will shed light on cytoskeletal mediated processes playing a major role in cellular activities such as cell division, morphogenesis, and functioning such as chloroplast positioning, pollen tube and root hair elongation and the movement of guard cells. Therefore, in the long run the screen described here has clear agricultural implications.